Project Summary: Integrins are transmembrane extracellular matrix (ECM) receptors composed of ? and ? subunits that form heterodimers. Integrins regulate many functions including cell adhesion and migration as well as signal and mechano-transduction. Integrins require both an ECM-binding ectodomain and a cytoplasmic tail that binds a variety of intracellular proteins to promote signaling. Integrins are implicated in tumor initiation, progression, and metastasis. However, integrin-targeted drugs, which focused on antagonizing the ectodomain, have largely failed in oncology clinical trials. These results suggest that the cytoplasmic tail may drive tumorigenesis independent of the ECM-binding ectodomain, though the mechanisms remain undefined. The principal integrins in the lung contain the b1 subunit. I have shown that integrin b1-knock out (KO) human lung adenocarcinoma cells injected into the lungs of athymic mice fail to form tumors. These data suggest that integrin b1 is required for lung adenocarcinoma development. Next, I developed integrin b1-KO cells that express either wildtype (WT) integrin b1, integrin b1 with a non-functional/mutated tail (b1 mt-tail) or integrin b1 with a non-functional ECM-binding ectodomain (Tac-b1). The WT and Tac-b1 cells formed soft agar colonies whereas the b1 mt-tail cells formed none. These data suggest that the integrin b1 tail is sufficient to permit tumor growth. RNA-seq gene expression analysis identified decreased epithelial-to-mesenchymal transition (EMT) and TGFb signaling signatures in integrin b1-KO cells. This correlation between integrin b1-KO and decreased expression of EMT-related genes was corroborated in human tumors. When the integrin b1-KO cells were further examined, I observed that while canonical TGFb signaling was unaffected, TGFb1-induced AKT phosphorylation was decreased. This observation is consistent with my mentor?s previous data that integrin b1 regulates AKT activation in a PI3K-independent manner that involves AKT lysine-63 polyubiquitination mediated by TRAF6. In summary, integrin b1 tail is required for tumor growth, and loss of integrin b1 is associated with loss of EMT and TGFb signal transduction. Based on these findings, I hypothesize that ECM-independent integrin b1 tail signaling regulates EMT and permits lung cancer growth by regulating TGFb1-induced AKT activation. I have developed two aims to test this hypothesis. In Aim 1, I will test the hypothesis that integrin b1 promotes tumor growth by facilitating EMT. I will use lung cancer mouse models and evaluate EMT in tumors with and without integrin b1 using histologic techniques and single cell RNA-seq. In Aim 2, I will test the hypothesis that the integrin b1 tail is sufficient for tumor development, and tail interactions with TRAF6 are required for TGFb-dependent AKT activation, EMT, and cell proliferation. I will use b1 mt-tail and Tac-b1 cells to define the role of the integrin b1 tail and the mechanisms whereby it facilitates TGFb signaling to promote lung cancer. Moving forward, I expect to leverage this training and data for R01-funding focused on integrin- targeted cancer therapeutics.